CN105431205A

CN105431205A - IgE antibodies for the inhibition of tumor metastasis

Info

Publication number: CN105431205A
Application number: CN201480033785.0A
Authority: CN
Inventors: 约瑟夫·A·莫利克; 张珀琳; 保罗·J·乌茨
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2013-06-12
Filing date: 2014-06-12
Publication date: 2016-03-23
Anticipated expiration: 2034-06-12
Also published as: US20140370001A1; EP3007773A4; CN105431205B; JP2016521750A; US9587032B2; US20170129965A1; US11390684B2; EP3007773B1; US20200087413A1; US10487152B2; JP6506745B2; EP3007773A1; WO2014201212A1

Abstract

The present invention provides novel IgE antibodies useful for inhibiting or preventing metastatic cancer. Also provided are methods to inhibit tumor metastasis by modulating the activity of at least one non-tumor cell, treating a patient to inhibit or prevent the appearance of tumor metastases derived from a primary solid tumor, treating established metastatic carcinoma, reducing metastasis of carcinoma cells, and reducing the growth kinetics of a primary solid tumor or a metastasized cell or tumor.

Description

The IgE antibody of Tumor suppression transfer

Related application

This application claims the U.S. Provisional Application No.61/834 submitted on June 12nd, 2013, the rights and interests of 169, and the U.S. Patent application No.13/939 that on July 11st, 2013 submits to, the rights and interests of 781.Whole instruction content whole of more than applying for are incorporated herein by reference.

Governmental support

Of the present invention making obtains governmental support, is specially the CA111639 contract of being subsidized by NIH (NationalInstitutesofHealth).Government enjoys some right in the present invention.

Background technology

IgE antibody mediation allergy and asthma reaction, features of these reactions are the super quick and inflammation delaying type response of anaphylactic type needing to raise effector lymphocyte.IgE antibody is transported to tissue from peripheral circulation, they can via the Fc receptor of following three types in conjunction with irritated effector lymphocyte there, such as mastocyte, basophilic granulocyte, eosinophilic granulocyte, dendritic cell, Langerhans cell, mononuclear cell and macrophage: Fc ε RI (or high-affinity Fc ε R) (K _a=10 ¹¹m ^-1), Fc ε RII (or low-affinity Fc ε R, CD23) (K _a<10 ⁸m ^-1) and hL-31.Be different from IgG antibody-like, IgE is attached to its FcR with high affinity, this affinity with regard to Fc ε RI than IgG to high about three orders of magnitude of the affinity of FcR (Fc γ RI-III), and with regard to Fc ε RII, with IgG to the affinity of its high-affinity Fc γ RI equally high (Gould, HJ, etal., Annu.Rev.Immunol., 21:579-628. (2003); Gounni, AS, etal., Nature, 367:183-186 (1994); Kinet, JP, Annu.Rev.Immunol., 17:931-72:931-972 (1999) and RavetchJV, andKinetJP, Annu.Rev.Immunol., 9:457-492 (1991)).

Irritated oncology (AllergoOncology) emerging field is based on showing the observed result and the research (TurnerMC that there is inverse correlation between the allergy that IgE mediates and cancer, etal., Int.J.Cancer, 118:3124-3132 (2006); WangH & DiepgenTL, Allergy60:1098-1111, (2005); TurnerMC, etal., Am.J.Epidemiol., 162:212-221 (2005); WangH, etal., Int.J.Cancer, 119:695-701 (2006); MillsPK, etal., Am.J.Epidemiol., 136:287-295 (1992); WiemelsJL, etal., CancerRes., 64:8468-8473 (2004); DodigS., etal., ActaPharm., 55:123-138 (2005) and WrenschM., etal., CancerRes., 66:4531-4541 (2006)).Therefore, the research worker in this field is exploring the treatment potentiality of IgE antibody class under following prerequisite in some cancer of prevention and therapy: may be useful when the immunoreation occurred as the adaptation response to microorganism/parasitic infection is at first at anti-malignant tumor.

The application of IgE in treatment of cancer advocates (Nagy by people such as Nagy, E., etal., CancerImmunol.Immunother., 34:63-69 (1991)), their major envelope glycoprotein (gp36) developed mouse mammary tumor virus (MMTV) has specific Mus IgE monoclonal antibody and confirms the remarkable anti-tumor activity (Nagy in the C3H/HeJ mouse with homogenic MMTV secreted adenocarcinoma of breast (H2712), E., etal., CancerImmunol.Immunother., 34:63-69 (1991)).The people such as Kershaw (Kershaw, MH, etal., Oncol.Res., 10:133-142 (1998)) develop the Mus monoclonal IgE of by name 30.6, and it is the epitopic specificity of expressing on the surface at Colon and rectum adenocarcinoma cell.Mice IgE30.6 inhibits the growth of the set Human colorectal carcinoma COLO205 cell of the subcutaneous growth in Reconstruction in Sever Combined Immunodeciency (SCID) mice, but this effect is of short duration.By contrast, mouse IgG 30.6 does not demonstrate Graft Versus Tumor.The certain effects of mice IgE is owing to the interaction between this antibody and the effector lymphocyte with Fc ε R, because this activity loses (Kershaw in particular by using unspecific mouse IgE in advance, MH, etal., Oncol.Res., 10:133-142 (1998)).The people such as Gould develop the specific mice/people of oophoroma tumor related antigen FBP (FBP) and are fitted together to IgE (MOv18-IgE) and IgGMOv18 (IgG1).In human ovarian cancer SCID mice xenograft model (IGROV1), the prolection of MOv18-IgE and MOv18-IgG1 is compared.The beneficial effect of MOv18-IgE be greater than MOv18-IgG1 and the persistent period longer, thus confirm the antitumous effect (Gould, HJ, etal., Eur.J.Immunol., 29:3527-3537 (1999)) of IgE antibody excellence.

Recently, the people such as Karagiannis confirm that mononuclear cell is by two kinds of different approach mediation IgE dependent tumors cell killings: ADCC (cytotoxicity of antibody dependent cellular mediation) and ADCP (phagocytosis that antibody dependent cellular mediates), these two kinds of approach mediate (Karagiannis by Fc ε RI and Fc ε RII, SN, etal., CancerImmunol.Immunother., 57:247-263 (2008) and Karagiannis, SN, etal., J.Immunol., 179:2832-2843 (2007)).This group also uses this Analytical system to confirm, and anti-Her2IgE can activated mononuclear cell and via ADCC killing tumor cell (KarragiannisP. in vitro, etal., CancerImmunol.Immunother., 58:915-930 (2009), on October 22nd, 2008 online publishing).Other example comprises the people such as Fu (Clin.Exp.Immunol., 153:401-409,2008), they demonstrated that the ADCC of IgE antibody-like mediation to inhibiting tumor cell be separated from Pancreas cancer patients, and the people (CancerImmunol.Immunother. such as Spillner, 61:1565-1573 (2012), they show use mononuclear cell, in vitro compared with anti-EGFRIgG, anti-EGFRIgE improves to the cytotoxicity of anti-human epithelial cancer cell line A431 up to 95%.

Although there is inspirer discovery in the emerging field of irritated oncology, still in the urgent need to the new therapeutic agent of exploitation treatment metastatic carcinoma.

Summary of the invention

The invention provides the novel I gE antibody that can be used for Tumor suppression growth and transfer.Provide the therapeutic monoclonal IgE antibody comprising people ε constant region and variable region of the binding specificity had tumor associated antigen (TAA).Additionally provide the method for the active reprogrammed at least one host source non-tumor cell being arranged in solid primary tumor microenvironment, thus reverse the ability that this non-tumor cell promotes growth and metastasis of tumours.Provide the metastatic carcinoma in treatment patient, suppress primary tumor that the method for the growth kinetics shifting and slow down constitutional or metastatic tumo(u)r occurs.

In one embodiment, the invention provides the method for the active reprogrammed of at least one host source non-tumor cell to the microenvironment being arranged in patient's solid tumor, wherein the ability of non-tumor cell mediate tumor transfer is suppressed, the method comprises the step using the specific IgE antibody of tumor associated antigen, wherein antibody forms ternary complex in the microenvironment of tumor, described ternary complex is by IgE antibody, tumor associated antigen and tumor environment endogenic host source non-tumor cell are formed, wherein antibody specificity is attached to tumor associated antigen and is positioned at the antibody receptor on the surface of host source non-tumor cell, this antibody receptor is that IgE is specific.

In another embodiment, the invention provides the method for the solid tumor transfer suppressed in patient, the method comprises uses the IgE antibody that can form ternary complex in the microenvironment of tumor to patient, described ternary complex is by the specific IgE antibody of tumor associated antigen, tumor associated antigen and tumor microenvironment endogenic host source non-tumor cell are formed, wherein antibody specificity is attached to tumor associated antigen and is positioned at the antibody receptor on the surface of host source non-tumor cell, this antibody receptor is that IgE is specific, and wherein after formation ternary complex, the ability of non-tumor cell mediate tumor transfer is suppressed.

Accompanying drawing explanation

Aforesaid and other target, feature and advantage can show more significantly from the description more specifically of following of the present invention preferred embodiment, as shown in drawings, in different views, and the parts that similar letter is corresponding identical.Accompanying drawing might not proportionally make, but is emphasizing to show principle of the present invention.

Fig. 1. the structure of chimeric IgE antibody and antigenic specificity.(A) figure of vector construct represents.Parent vector is pcDNA3.1/ neomycin (pEpsilonI), pEF6/ blasticidin-S (pEpsilonII), pcDNA3.1/ bleomycin (pKappaI) and pcDNA3.1/ hygromycin (pKappaII).Heavy chain and the chain variable region gene hybridoma clone from table 1, and insert the upstream of people ε or people k constant region gene.(B) SDS-PAGE analyzes.Be separated at 4-12% gradient acrylamide gel by electrophoresis from the chimeric IgE of the CHO-K1 clone purification of transfection and the contrast people IgE that derives from myeloma cell line SKO-007, and pass through Coomassie blue stain.(C) Antibody specificity analyses.Superposition block diagram shows compared with the cell line of untransfected, is attached to the facs analysis of the IgE of the cell line with isoantigen transfection.1F5.hIgE (anti-hCD20) combines the A20 cell of employment CD20cDNA transfection, and 3C6.hIgE (anti-hMUC1, the form of partial glycosylation) detects people MUC1 on the mouse mammary carcinoma cell line 4T-1 with hMUC1cDNA transfection.Broken line graph is shown and is detected the ELISA of the hMUC1 tandem repeat peptide (50mer) of synthesis by 4H5.hIgE (anti-hMUC1 peptide backbone).Control peptide is derived from second born of the same parents' outer shroud (43mer) of hCD20.

Fig. 2. the tumor cell in vitro toxicity of mastocyte and eosinophilic granulocyte's mediation.Block diagram display %PI ⁺cFSE ⁺tumor cell.By OCI-Ly8 human B cell lymphoma cell (hCD20 ⁺) with 10 ^-5mMCFSE labelling 15 minutes.By 1 × 10 ⁵the cell of individual labelling and undyed CBMC and 2.5 μ g/mlIgE (A and B) or umbilical cord haematogenous eosinophilic granulocyte (CBEo) and 5 μ g/mlIgE (C and D) mix, then at 37 DEG C of incubation 24h.By cell mixture propidium iodide (PI) dyeing, then carry out flow cytometry.PI ⁺cell is at CFSE ^hipercentage ratio in component represents total cytotoxicity, and result illustrates with bar graph form.(A) different effector lymphocytes: the cytotoxicity of CBMC and hIgE mediation under target cell ratio.(B) blocking antibody or the isotype controls (E:T ratio 4:1) of 2 μ g/ml is added.(C) different effector lymphocytes: the cytotoxicity of CBEo and hIgE mediation under target cell ratio.(D) 2 μ g/ml blocking antibodies or 10U/ml heparin is added.(E:T ratio 2.5:1).Here also show by the data of the tumor mortality of independent antibody induction (that is, when there is not effector lymphocyte).The result of display is the meansigma methods ± SD of a representative experiment.Student t checks *, p<0.05; *, p<0.01; * *, p<0.005.

Fig. 3. tumour-specific IgE suppresses tumor growth in vivo.At the 0th day by 10 ⁵individual 4T1.hMUC1 cancer cell subcutaneous is inoculated into the both sides of hFc ε RI mice.Used 20 μ g to contrast or 3C6.hIgE (anti-hMUC1) at the 1st, 2,3,4 and 5 day.Obtain 2 dimension kind of calliper values, until tumor area is more than 300mm ².(A) average tumor size is to the curve chart of time.Error bars represents the meansigma methods ± SD of each group.Point out in bracket in survival mice/total number of mice of last time point each group.Anti-hMUC1 group is obviously different from matched group (two-way ANOVA, p<0.001).(B and C) from the experiment shown in (A) in the 34th day results of the mice from survival tumor, section dyeing for mastocyte.Mastocyte does not exist substantially in the center of tumor and in all regions of tumor.Mastocyte (red arrow) the non-threshing (B) existed.Only there is a tumor of the mice from 3C6.hIgE processed group to contain by the region of mast cells infiltration, which show the evidence (C) of threshing.

Fig. 4 .hMUC1 specific mice IgE mediates the complete tumor rejection in Fc ε RI.By four kinds of various combinations of the 4T1 tumor cell of employment MUC1, anti-hMUC1IgE (3C6.mIgE) and/or cytokine (MCP-1, IL-5) transfection subcutaneous vaccination in the 0th day to hFc ε RITg ⁺the both sides of mice.Every mice uses 10 altogether ⁵individual cell (n=4/group).(A) explanation of four experimental grouies.(B) experiment be designed to test I gE antibody and isoantigen (MUC1) and by the interaction of myeloid cell that attracts of two kinds of cytokines studying.By mixing with cells, be then subcutaneously injected into the both sides (100,000 cell/mice) of Fc ε RIKO/Tg mice immediately.Tumor growth is monitored by two-dimentional slide calliper rule growth.Except the 4th group, in each group, all observed progressive growth, in the 4th group, tumor can temporary stereognosis, then permanent regression.Shown data represent two independent experiments.Error bars represents average tumor size ± SD.

Fig. 5 .hMUC1 specific mice IgE mediates the complete tumor rejection in Fc ε RIKO/Tg mice.Each growth curve and derive from the measurement of tumor meansigma methods repeating to test of experiment shown in Fig. 6.The 4T1 tumor cell of expressing 3C6.mIgE or cytokine is mixed as shown in FIG.Data derive from the meansigma methods (B, D) of each measurement of tumor (A, C) or gross tumor volume.Note, repeating in experiment to there are one group of totally 4 tumor showing growth, although growth kinetics reduces greatly.

The specific mice IgE of Fig. 6 .hMUC1 can not mediate tumor rejection completely in wild-type mice, but discloses the ability of prevention transfer.The each group of 4T1 tumor cell of expressing 3C6.hIgE or cytokine is mixed as shown in FIG, and injects Balb/c mice.Show each tumor growth curve (A, B), and the meansigma methods (C, D) of group.The 4th group of tumor do not grown in Fc ε RIKO/Tg mice grows, although growth kinetics reduces greatly in wild-type mice.Note, the 3rd group of tumor (independent 4T1/hMUC1 and cytokine) shows medium growth kinetics.Three Fc ε RIKO/Tg transgenic mices comprise in this experiment as positive control, and all three with all failing to show any tumor growth (D schemes,--) during the 4th group of tumor injection.The growth kinetics that 3rd group of tumor (only MUC1 and cytokine) is reduced may come from the following fact: these mices there occurs transfer in early days tumor development process, and start lose weight (Fig. 7) to when the 10th day.The mice with the 4th group of tumor (3C6-mIgE+MUC1+ cytokine) does not demonstrate and to lose weight or other significantly shift sign, although have little tumor in its both sides.

Fig. 7. the kinetics that loses weight in the 3rd and the 4th group of mice.Within every 5 days, weigh to the wild-type mice of the 3rd and the 4th group of tumor injection, and calculating mean value and standard deviation.Note, the notable difference of average weight can be detected by the 10th day between the two groups.Mouse Weight in 3rd group alleviates fast, produce ascites and the perpendicular hair of appearance.These whole bodies transfer sign does not all occur in the wild-type mice with the 4th group of tumor.

Detailed description of the invention

The invention provides the novel I gE antibody that can be used for suppressing or prophylaxis of tumours grows and shifts.Neoplasm metastasis partly by bone marrow (marrow sample) source cell of the cytokine be present in tumor microenvironment and innate immune system interaction and drive.Such as marrow sample T suppression cell is attracted in tumor microenvironment by the local expression of tumor cell to cytokine, then causes these cells to discharge other cytokine.Therefore cytokine environment in tumor microenvironment becomes complex by tens even hundreds of cytokines and chemotactic factor, and these factors facilitate cancer related inflammation.

Be correlated with marrow source sexual cell such as macrophage and mastocyte of the tumor accumulated in tumor microenvironment seems (DePalmaetal., CancerCell23 (3): 277-286 (2013) relevant to tumour progression; Daltonetal., CancerImmunolImmunotherDOI10.1007/s00262-012-1246-0, on April 18th, 2012 online publishing).This association regulates and controls by the complex cell cytokine environment existed in tumor microenvironment.Such as, Metastasis in Breast Cancer strengthens (RuffellBetal., TrendsImmunol.33:119 (2012)) when existence 2 cytokines by macrophage.In addition, in response to cytokine, other cells are mononuclear cell, granulocyte and mastocyte secretory protein hydrolytic enzyme such as, these enzyme extracellular matrixes (ECM) are modified, thus cause discharging the ECM binding growth factor (HanahanD & CoussensLM, the CancerCell21:309 (2012) that promote transfer; LuP, etal., ColdSpringHarb.Perspect.Biol., 3:a005058 (2011)).Therefore, it seems " myeloid cell of the chronic activation in tumor tissues supports many tumoral character " (CoussensLMetal., Science339:286-291 (2013)); (HanahanD & CoussensLM, CancerCell21:309 (2012)).

The present inventor have been surprisingly found that: antibody of the present invention can suppress the transfer of solid primary tumor and secondary tumors and higher level tumor.Be not bound by theory, can to the host source non-tumor cell reprogrammed in the tumor microenvironment of primary tumor according to IgE antibody of the present invention, shift to make Tumor suppression or the generation of prophylaxis of tumours transfer.The present invention is unique and beat all, because it carrys out the behavior of regulate tumor cell by intermediate host source cell.Final result is that primary tumor can not shift.This with record in a large number and each side of the well-known cancer therapy based on antibody is far from each other, based in the cancer therapy of antibody, the phagocytosis (ADCP) of the cytotoxicity (ADCC) of antibody dependent cellular mediation and antibody dependent cellular mediation is adopted to carry out Therapeutic cancer directly to kill cancerous cell (Fu, etal.Clin.Exp.Immunol., 153:401-409,2008; KaragiannisP., etal., CancerImmunol.Immunother., 58:915-930 (2009), on October 22nd, 2008 online publishing; Karagiannis, SN, etal., CancerImmunol.Immunother., 57:247-263 (2008) and Karagiannis, SN, etal., J.Immunol., 179:2832-2843 (2007)).

In one embodiment of the invention, non-tumor cell such as marrow sample T suppression cell reprogrammed in host source is carried out in the tumor microenvironment of solid primary tumor.The reprogrammed of host source non-tumor cell is such as mediated by the cytokine in described tumor microenvironment and tumour-specific IgE antibody after the tumour-specific IgE antibody using effective dose to experimenter.In one embodiment, reprogrammed is included in tumor microenvironment and forms ternary complex.In one embodiment, this ternary complex in tumor microenvironment, by IgE antibody, has the cell such as tumor cell or solubility tumor related antigen of tumor associated antigen in its surface, and host source non-tumor cell is formed.After the antibody using effective dose to experimenter, antibody specificity is attached to tumor associated antigen and is positioned at the antibody receptor on non-tumor cell surface, host source.

Term " tumor microenvironment " or " mesenchyma stroma of tumors " mean around tumor cell and support the tissue of tumor cell, cell, molecule and blood vessel.The microenvironment of tumor is dynamic, and tumor can change its microenvironment, and microenvironment can affect the mode of tumor growth and diffusion.

In one embodiment, host source non-tumor cell is the endogenous resident cells of tumor microenvironment.As used herein, one or more cells that " tumor microenvironment is endogenic " that use means to insert the dynamic compartment (dynamiccompartment) in tumor nest (tumornest) or around the cell type of tumor nest can be exchanged with term " mesenchyma stroma of tumors is endogenic ", and include but not limited to the connective tissue of time to time change, vascular and inflammatory cell, and comprise tumor cell itself.Other host source non-tumor cells of mesenchyma stroma of tumors comprise fibroblast and vascular cell, include but not limited to mesenchyme source cell (such as fibroblast, vascular progenitor, endotheliocyte, adipose cell and precursor thereof and vascular endothelial cell) and not isophenic marrow source sexual cell, include but not limited to mastocyte, basophilic granulocyte, mononuclear cell, macrophage, eosinophilic granulocyte, neutrophil cell, dendritic cell, Langerhans cell, platelet and CFU-GM thereof and infiltrating lymphocytes, comprise CD4 and cd8 t cell and B cell.Irritated effector lymphocyte and/or marrow sample T suppression cell are also referred to as marrow source sexual cell or myeloid cell herein.

In a preferred embodiment, antibody comprises people Fc ε constant region.In a preferred embodiment, IgE antibody is CA125, FBP (FBP), HER2/neu, MUC1 and PSA specific antibody.

As used herein, " experimenter " is human patients or other animals, such as another kind of mammal, it has functional mast cell, basophilic granulocyte, neutrophil cell, eosinophilic granulocyte, mononuclear cell, macrophage, dendritic cell and Langerhans cell.In people, all these cells all express the high-cutting slope along road (Fc ε RI) of used IgE antibody of the present invention.

In some cases, IgE antibody of the present invention can be used for suppressing the diffusion (also referred to as shifting) from primary tumor to the position different from the position of primary tumor.These other tumor locus when the result for shifting from primary tumor site sometimes referred to as Secondary cases, three property, four property or higher level tumor locus.Primary tumor release cycle cancerous cell, first these cancerous cell must set up microenvironment in various tissue and organ such as liver, lung and skeleton.These finally produce and significantly shift clinically with on radiograph.Be different from the IgG antibody mainly stayed as its of part in lacuna vasorum, IgE antibody is moved out of vascular space and is arrived each tissue in body intrinsic biology.

In one embodiment, tumor microenvironment endogenic host source non-tumor cell reprogrammed is carried out in the microenvironment of the Secondary cases formed by circulating cancer cells, three property, four property or higher level tumor locus.In this second embodiment, cell reprogrammed, by the tumor specific antibody mediation in described Secondary cases, three property, four property or higher level tumor microenvironments, wherein uses the tumor specific antibody of effective dose to experimenter.

In one embodiment, host source non-tumor cell is tumor microenvironment endogenic marrow source property T suppression cell.In a preferred embodiment, property T suppression cell in marrow source is independently selected from mastocyte, basophilic granulocyte, neutrophil cell, eosinophilic granulocyte, mononuclear cell, macrophage, dendritic cell and Langerhans cell.In one embodiment, host source non-tumor cell is mesenchyme source cell, and independently selected from fibroblast, vascular progenitor, endotheliocyte, adipose cell and precursor thereof.

The present inventor has also found that antibody of the present invention can repel by mediate tumor.It is confirmed that, the marrow source sexual cell mediation of this effect by being activated by IgE and Tumor-associated cell surface antigen.Killing and wounding of tumor is the result of following aspect: the local effect of IgE antibody, exists marrow source sexual cell in environment, and macrophage, Eosinophil Chemotaxis's cytokine.In one embodiment, IgE antibody of the present invention be used in and shift after at Secondary cases, three property, four property or higher level microenvironment position inducing death of neoplastic cells.In this second embodiment, death of neoplastic cells at Secondary cases, three property, four property or higher level microenvironment position is by changing the pattern of the cytokine wherein existed and changing the state of activation of non-tumor cell and mediate, and all these are all in response to the tumor specific antibody in described microenvironment.In a preferred embodiment, death of neoplastic cells needs to form ternary complex in described microenvironment, and this ternary complex is made up of IgE antibody, the tumor cell with target antigen and non-tumor cell.Specifically, IgE antibody is specifically bound to the antigen be positioned on tumor cell surface and the antibody receptor be positioned on non-tumor cell surface, wherein uses the antibody of effective dose to experimenter.In another embodiment, non-tumor cell is marrow source property T suppression cell.In a preferred embodiment, property T suppression cell in marrow source is independently selected from mastocyte, basophilic granulocyte, neutrophil cell, eosinophilic granulocyte, mononuclear cell, macrophage, dendritic cell and Langerhans cell.In a preferred embodiment, IgE antibody is anti-MUC1IgE.

The present inventor finds further: antibody of the present invention can be used for slowing down solid primary tumor or transitional cell or the metastatic tumor growth kinetics in patient body.In one embodiment, marrow source sexual cell reprogrammed is carried out in the tumor microenvironment of solid primary tumor or one group of transitional cell.In this second embodiment, reprogrammed, by the tumor specific antibody mediation in described tumor microenvironment, wherein uses the tumor specific antibody of effective dose to experimenter.In a preferred embodiment, reprogrammed needs to form ternary complex in solid tumor or transitional cell or metastatic tumor, described ternary complex is made up of the tumor cell of antibody, solid tumor or transitional cell or metastatic tumor and non-tumor cell, wherein antibody specificity is attached to the antigen be positioned on tumor cell surface and the antibody receptor be positioned on non-tumor cell surface, and wherein uses the antibody of effective dose to experimenter.In another embodiment, non-tumor cell is marrow source sexual cell.In a preferred embodiment, marrow source sexual cell is the myeloid cell independently selected from mastocyte, myeloblast, basophilic granulocyte, neutrophil cell, eosinophilic granulocyte, mononuclear cell, macrophage, dendritic cell and Langerhans cell.In a preferred embodiment, antibody is IgE.In a preferred embodiment, IgE antibody is anti-MUC1IgE.As used herein, " experimenter " is human experimenter or other animals with functional mast cell, myeloblast, basophilic granulocyte, neutrophil cell, eosinophilic granulocyte, mononuclear cell, macrophage, dendritic cell and Langerhans cell, and these cells have receptor affinity to used IgE antibody of the present invention.

The growth kinetics of solid primary tumor as used herein or transitional cell or metastatic tumor slows down or eliminates the implication being defined as meaning to understand this area completely.Such as, slowing down of growth kinetics means: for given tumor type, the exponential growth of solid primary tumor, transitional cell or metastatic tumor, specific growth rate or doubling time relative in vivo or external exponential growth, specific growth rate or the doubling time usually observed reduce.Eliminating completely of tumor in symptom, by health check-up or radiophotography, finds to there is not tumor when there is IgE antibody, wherein finding to there is tumor before by these detection methods.

" therapeutic IgE antibody " of the present invention (herein also referred to as " monoclonal IgE antibody of the present invention ") is the monoclonal antibody comprising people epsilon (ε) constant region and comprise variable region, described variable region comprises at least one antigen binding domain, described antigen binding domain is that tumor associated antigen (TAA) is specific, and described tumor associated antigen is be arranged in the cell surface antigen or Soluble cancer antigen that tumor microenvironment is close to the tumor of carrying out treating in other words conj.or perhaps.It is generally acknowledged, the therapeutic dose of IgE antibody of the present invention is by far below IgE class (such as, the Herceptin with anticancrin therapy and Rituximab ) relevant dosage, this is not only because IgE is to the high-affinity of Fc ε RI, also because method of the present invention comprises and carries out reprogrammed to tumor microenvironment one or more host source non-tumor cells endogenic.IgE antibody seems to have cascading in tumor environment, and this promotes the suppression to the transfer of solid tumor.This cascading means: do not need according to IgE antibody of the present invention to use the pharmacodynamics effect mediated target antigen, and therefore such as do not need to make CD20 or HER2/neu receptor saturated, such as, this monoclonal cancer therapy based on IgG for routine is then required.IgE antibody active transport goes out vascular space, is attached to the fc ε R shown on myeloid cell precursor.This and IgG antibody form sharp contrast, and the latter is usually located in vascular space, and leave this space by means of only mass action through the blood vessel that there occurs Penetration ration change.

As the term is employed herein " tumor associated antigen " (TAA) can be any type known in the art can be relevant to tumor cancer antigen, and comprise and be present in antigen on cell surface (comprising tumor cell) and Soluble cancer antigen.Various kinds of cell surface antigen on tumor and normal cell has solubility homologue.Such antigen includes but not limited to be present in those antigens in carcinoma-associated fibroblasts (CAF), tumor endothelial cell (TEC) and tumor-associated macrophages (TAM).The example of carcinoma-associated fibroblasts (CAFs) target antigen includes but not limited to: carbonic anhydrase IX (CAIX), fibroblast activation protein alpha (FAP α) and matrix metalloproteinase (MMP), comprise MMP-2 and MMP-9.The example of tumor endothelial cell (TEC) target antigen includes but not limited to VEGF (VEGF), comprises VEGFR-1,2 and 3; CD-105 (Endothelin); Tumor endothelial marker (TEM), comprises TEM1 and TEM8; MMP-2; Survivin; With prostate specific membrane antigen (PMSA).The example of tumor-associated macrophages antigen includes but not limited to: CD105, MMP-9, VEGFR-1, VEGFR-2, VEGFR-3 and TEM8.

In one embodiment, therapeutic IgE antibody can for being positioned at cancer antigen such as VEGFR-2, MMP on non-tumor cell, survivin, TEM8 and PMSA are specific.Tumor antigen can be epithelial cancer antigen (such as mammary gland, gastrointestinal, lung), prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), bladder cancer antigen, lung (such as minicell lung) cancer antigen, colon cancer antigen, ovarian cancer antigen, brain cancer antigen, gastric cancer antigen, renal cell carcinoma antigen, pancreatic carcinoma antigen, hepatocellular carcinoma antigen, esophageal carcinoma antigen or head and neck cancer antigen.Cancer antigen can also be lymphoma antigen (such as, non-Hodgkin lymphoma or Hodgkin lymphoma), B cell lymphoma cancer antigen, leukemia antigen, myeloma (that is, multiple myeloma or plasma cell myeloma) antigen, acute lymphoblastic leukemia antigen, chronic myelogenous leukemia antigen or acute myelogenous leukemia antigen.

Other cancer antigens include but not limited to be present in the Mucin1 albumen on most people adenocarcinoid or peptide (MUC-1): pancreas, colon, mammary gland, ovary, lung, prostate, head and neck, comprise multiple myeloma and some B cell lymphomas; Human epidermal growth factor receptor-2 (HER-2/neu) antigen; EGF-R ELISA (EGFR) antigen relevant to pulmonary carcinoma, head and neck cancer, colon cancer, breast carcinoma, carcinoma of prostate, gastric cancer, ovarian cancer, the brain cancer and bladder cancer; The prostate specific antigen (PSA) generally expressed in Androgen Independent Prostate Cancer and/or prostate specific membrane antigen (PSMA); The gp-100 (glycoprotein 100) relevant to melanoma cancer embryo (CEA) antigen; With Lewis (Lewis) blood group A substance about and the carbohydrate antigen 19.9 (CA19.9) relevant with colorectal cancer; And melanoma cancer antigen, such as MART-1.

Other antigens comprise mesothelin, FBP (FBP), CA125 (CA-125) and melanoma associated antigen, such as NYESO1.

In one embodiment, cancer antigen is release, the soluble form of cell surface cancer antigen.In a preferred embodiment, tumor associated target antigens is strictly for being positioned at the cell surface antigen on tumor cell surface.In a preferred embodiment, tumor associated antigen is selected from CA125, FBP (FBP), HER2/neu, MUC1 and PSA.

" monoclonal antibody " refers to the prepared product of single molecular antibody as the term is employed herein.Monoclonal antibody combination goes out single binding specificity and affinity for specific epitope display.Monoclonal antibody of the present invention is preferably chimeric, humanized or total man, so that when experimenter host behaves in conjunction with people Fc epsilon receptor.Humanization and human antibody are also used in the immunogenicity of reduction to the Mus component of such as chimeric antibody when host subject behaves.Monoclonal antibody is by including but not limited to prepared by the standard technique of recombinating and synthesizing.

Term " chimeric mAb " refers to the antibody showing single binding specificity, and it has and is one or morely derived from the district of an antibody and one or more district being derived from another antibody.In one embodiment of the invention, constant region is derived from people epsilon (ε) constant region (heavy chain) and people k or λ (light chain) constant region.The variable region of chimeric IgE monoclonal antibody of the present invention is generally inhuman source, is such as derived from rodent, such as mice (Mus), rabbit, rat or hamster.

As used herein, " humanization " monoclonal antibody comprises the constant region being derived from people ε constant region (heavy chain) and people k or λ (light chain) constant region.The variable region of antibody preferably comprises people source skeleton and non-human antigen land (CDR).

Total man or human-like antibodies produce by carrying out vaccination to genetic engineering modified animal, described animal is such as at Chinese mugwort uncle gene Coase (Abgenix) company (ThousandOaks, and this (MedaRex) (Princeton of wheat Derek CA), NJ) mouse species produced, it contains human normal immunoglobulin heredity storehouse and produces human antibody in response to vaccination.In addition, use phage display library, the coding region of the people variable region that can identify in antigen selection algoscopy and select is incorporated to, to produce the human normal immunoglobulin variable region in conjunction with target antigen.

Term " antigen binding domain " refers to that antibody of the present invention contains with the amino acid residue of AI also for antibody gives its specificity to antigen and the part of affinity.Antibody district comprises correct ways necessary " skeleton " amino acid residue maintaining antigen binding residues.

" antigen " is the part of molecule or the molecule that can be selectively bound by the antibody, its can also induced animal produce can in conjunction with the antibody of the epi-position of this antigen.Antigen can have one or more identical or different epi-position.In a preferred embodiment, antibody of the present invention is single epitope specificity.In one embodiment, antigen can be combined by IgE antibody of the present invention, and to form immune complex, this immune complex combines with marrow sample effector lymphocyte can to tumor microenvironment reprogrammed to suppress or prophylaxis of tumours grows or shifts.In one embodiment, antigen in itself for a variety of reasons may not immune response stimulating, these reasons are such as antigen is " self " antigen, usually be not that needs are reacted by immune system recognition, or otherwise immune system become the tolerance of this antigen and does not produce immunoreation.In another embodiment, antigen is MUC1.

Term " epi-position " means the part that antigen can be selectively bound by the antibody by antibody recognition and at one or more places of antigen-binding site.Epi-position comprises the chemically reactive surface group of molecule usually, such as aminoacid or sugared side chain, and has specific three dimensional structure characteristic and specific charge characteristic.In one embodiment, the epi-position of antigen is repeated epi-position.In one embodiment, the epi-position of antigen is non-repeatability epi-position.

" ternary complex " is the complex formed in tumor microenvironment, it is made up of IgE antibody of the present invention, cell surface tumor associated target antigens and host source non-tumor cell, and wherein antibody specificity is attached to tumor associated antigen and is positioned at the antibody receptor on non-tumor cell surface.

In a preferred embodiment, antigen is CA-125, FBP (FBP), HER2/neu, MUC1 or PSA.In one embodiment, non-tumor cell is effector lymphocyte.In one embodiment, effector lymphocyte is medullary system allergy and/or parasiticide effector lymphocyte.In a preferred embodiment, IgE is anti-MUC1IgE.In a preferred embodiment, antibody receptor is Fc ε RI.

In one embodiment, ternary complex comprises the IgE antibody being attached to Soluble cancer antigen He being positioned at the antibody receptor on non-tumor cell surface, host source.In a preferred embodiment, ternary complex comprises the tumor associated target antigens being attached to and expressing on tumor cell surface and the IgE antibody being positioned at the Fc ε RI on the non-tumor cell of tumor microenvironment endogenic host source.

Promote antibody (such as to the murine antibody of antigen) and determine that the method whether selected antibody is attached to unique epitope is well known in the art.

Antibody needed for screening completes by technology known in the art, these technology are such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometry, GDP reacts, immunodiffusion assays, original position immunoassay (adopts such as gold colloidal, enzyme or labelled with radioisotope), western (Western) trace, precipitation, agglutination assay (such as gel agglutination assays, blood clotting algoscopy), complement fixation algoscopy, immunofluorescence assay, protein A algoscopy and immunoelectro-phoresis assays etc.Many modes known in the art are used for detecting in immunoassay combining, and these modes within the scope of the invention.

For the preparation of monoclonal antibody, can use and produce any technology of antibody molecule (see such as Antibodies--ALaboratoryManual by the continuous cell line in cultivation, HarlowandLane, eds., ColdSpringHarborLaboratoryPress:ColdSpringHarbor, NewYork, 1988).These include but not limited to initial by Kohler and Milstein (1975, Nature256:495-497) hybridoma technology developed, and three tumor (trioma) technology, human B-lymphocyte hybridoma technology (Kozboretal., 1983, ImmunologyToday, 4:72) and EBV-hybridoma technology with human monoclonal antibodies (Coleetal., 1985, inMonoclonalAntibodiesandCancerTherapy, AlanR.Liss, Inc., pp.77-96).In other embodiments of the present invention, up-to-date technology (PCT/US90/02545) can be adopted in germfree animal to produce monoclonal antibody.According to the present invention, can end user's antibody, and people's antibody is by end user's hybridoma (Coteetal., 1983, Proc.Natl.Acad.Sci.U.S.A., 80:2026-2030) or by using EBV virus Transformation human B cell (Coleetal. in vitro, 1985, inMonoclonalAntibodiesandCancerTherapy, AlanR.Liss, pp.77-96) and obtain.In fact, according to the present invention, can use by by the gene of the amouse antibody molecule from polypeptid specificity and the technology (Morrisonetal. producing " chimeric " antibody from the gene splicing with suitable bioactive human antibody molecules and develop together, 1984, J.Bacteriol.159:870; Neubergeretal., 1984, Nature312:604-608; Takedaetal., 1985, Nature314:452-454); Such antibody within the scope of the invention.

In one embodiment, antibody of the present invention is IgE monoclonal antibody, it contains and is selected from following nucleotide sequence: by comprise SEQIDNO:1 nucleic acid sequence encoding variable region of heavy chain, by the variable region of light chain of nucleic acid sequence encoding and the combination in any thereof that comprise SEQIDNO:2, and wherein heavy chain and light chain are grafted on people ε heavy chain and k light chain gene respectively.

In one embodiment, antibody of the present invention is IgE monoclonal antibody, it contains and is selected from following nucleotide sequence: the variable region of light chain of the variable region of heavy chain by the nucleic acid coding of SEQIDNO:3, the nucleic acid coding by SEQIDNO:4 and combination in any thereof, and wherein heavy chain and light chain are grafted on people ε heavy chain and k light chain gene respectively.

In one embodiment, the invention provides monoclonal antibody 3C6.hIgE, its comprise from VU-3C6 hybridoma clone's and be grafted to the variable region of IgG light chain people Igk light chain and ε heavy chain gene and heavy chain respectively.The mucinous high glycosylation form of the process LAN of VU-3C6 targeted human MUC-1 (hMUC1)-in the tumor produced by glandular epithelium.In one embodiment, the present invention includes IgE antibody 4H5hIgE, it is that the different MUC1 isotype of MUC1 isotype specific from 3C6.hIgE is specific.

In one embodiment, antibody of the present invention is monoclonal antibody 3C6.hIgE, and it contains by the variable region of heavy chain of the nucleic acid sequence encoding comprising SEQIDNO:1, by the variable region of light chain of nucleic acid sequence encoding comprising SEQIDNO:2.

In one embodiment, antibody of the present invention is monoclonal antibody 4H5hIgE.Antibody 4H5.hIgE has the variable region of light chain of the variable region of heavy chain by the nucleic acid coding of SEQIDNO:3 and the nucleic acid coding by SEQIDNO:4, and these districts are grafted on people Igk light chain and ε heavy chain gene.

In one embodiment, antibody of the present invention is the IgE monoclonal antibody of the epitope specificity of MUC1.In one embodiment, antibody of the present invention is the epitope specificity of MUC1, and described epi-position comprises the aminoacid STAPPAHGVTSAPDTRPAPG [SEQIDNO:5] of MUC1.Definite epi-position is arranged in one of 20 the aminoacid repetitions characterizing MUC1 outer domains.In one embodiment, the antibody of the present invention epi-position place that can limit at STAPPAHGVTSAPDTRPAPG [SEQIDNO:5] is in conjunction with MUC1.

In one embodiment, antibody according to the present invention is expressed by Positive transfections tumor, and described transfectoma is by enzyme-linked immunosorbent assay (ELISA) and western (Western) trace qualification.Positive transfections tumor will be cloned by limiting dilution for maximum output, and produce for antibody and select.As used herein, " transfectoma " comprises the restructuring eukaryotic host cell of expressing antibody, such as Chinese hamster ovary (CHO) cell and NS/O myeloma cell.Such transfectoma method is (Morrison, S. (1985) Science, 229:1202) well known in the art.That announces to be fitted together to or the method for humanized antibody successfully creates various people's chimeric antibody or antibody fusion protein (HelgueraG for generating mice/people before, PenichetML., MethodsMol.Med. (2005) 109:347-74).

In general, gomphosis mouse-human monoclonal antibodies (that is, chimeric antibody) produces by recombinant DNA technology known in the art.Such as, the gene digestion with restriction enzyme of the Fc constant region of Mus of encoding (or other species) monoclonal antibody, to remove the district of coding Mus Fc, and substitutes into the equal parts of the gene of encoding human Fc constant region.(see Robinsonetal., international patent publications PCT/US86/02269; Akira, etal., european patent application 184,187; Taniguchi, M., european patent application 171,496; Morrisonetal., european patent application 173,494; Neubergeretal., international application WO86/01533; Cabillyetal. U.S. Patent No. 4,816,567; Cabillyetal., european patent application 125,023; Betteretal. (1988Science, 240:1041-1043); Liuetal. (1987) PNAS, 84:3439-3443; Liuetal., 1987, J.Immunol., 139:3521-3526; Sunetal. (1987) PNAS, 84:214-218; Nishimuraetal., 1987, Canc.Res., 47:999-1005; Woodetal. (1985) Nature, 314:446-449 and Shawetal., 1988, J.NatlCancerInst., 80:1553-1559).

Chimeric antibody is by with the further humanization of under type: the sequence of the Fv variable region be not directly involved in antigen combines replaced with the equivalent sequence from mankind Fv variable region.Morrison, S.L., 1985, Science, 229:1202-1207 and Oietal., 1986, BioTechniques, 4:214 provide the generality summary of humanized chimeric antibody.These methods comprise separation, handle and express all or part of nucleotide sequence of the IgF v variable region in encoding heavy chain or light chain at least one.The source of such nucleic acid is well known to those skilled in the art, and such as can derive from 7E3, and this is the anti-GPII of a kind of generation _biII _athe hybridoma of antibody.Then the recombinant DNA of encoding chimeric antibodies or its fragment can be cloned into suitable expression vector.Suitable humanized antibody replaces by CDR alternatively and produces (U.S. Patent No. 5,225,539; Jonesetal.1986Nature, 321:552-525; Verhoeyanetal.1988Science, 239:1534 and Beidleretal.1988J.Immunol., 141:4053-4060).

In one embodiment, use various strategy, be minimized compared with making the immunogenicity of IgE monoclonal antibody of the present invention and such as drawing the parental generation antibody of this antibody.Such as, by making the immunogenicity of chimeric IgE monoclonal antibody to people experimenter of the people of comprising Fc ε constant region of the present invention and Mus variable region lower with under type: carry out genetic engineering modified to the humanized antibody comprising constant region and variable region, described constant region is derived from people Fc ε, described variable region comprises people source skeleton and non-human antigen land, and described antigen binding domain maintains the antigenic specificity identical with parental generation chimeric antibody.Alternatively, comprising the total man that is fitted together to the identical antigenic specificity of IgE monoclonal antibody with parental generation or human-like antibodies, also known program can be used to carry out genetic engineering modified.

Immunogenic additive method for reducing IgE monoclonal antibody of the present invention includes but not limited to such as DE-IMMUNIZATION ^tMthe method of (BiovationLtd., Aberdeen, UnitedKingdom and MerckKgaA, Darmstadt, Germany).This technology is such process, and the qualification of this process can cause the upper Mus epi-position existed of immunogenic Mus or chimeric mAb such as " human anti-mouse antibody " (HAMA) or " the anti-chimeric antibody of people " (HACA) in the mankind.This process also changes these epi-positions to avoid compared with the antibody not accepting this process or at least to reduce immunogenicity in heredity.

Reduce the immunogenic additive method (Lazaretal. of monoclonal antibody, MolImmunol., 44,1986-1998 (2007)) qualification can activate helper T cell thus the skeleton causing HAMA or HACA to react and antigen binding domain peptide or conformation motif.Use the method, new quantitative normal form is used for " humanized " that determine Mus variable region, and the Mus district of low humanized is replaced to the district of higher humanized, thus reduce the immunogenicity of antibody.

As used herein, induction IgE mediation immunoreation comprise following one or more:

I) in tumor microenvironment or on tumor cell, form ternary complex, it comprises and IgE antibody is attached to marrow source cell and is attached to tumor specific antigen, to make to suppress or prophylaxis of tumours transfer the reprogrammed of marrow source cell;

Ii) super quick type reaction, it is stimulated by the antigen especially in tumor microenvironment/IgE immune complex, as by the mastocyte of immune complex being attached to via IgE antibody receptor Fc ε RI and/or Fc ε RII and the threshing of basophilic granulocyte thus cause such as discharging histidine confirm;

Iii) the direct targets neoplastic cells via the ADCC immunoreation of the antigen/IgE immune complex of antagonism especially in tumor microenvironment, ADCP immunoreation or ADCC and ADCP two kinds of immunoreation, as when being attached to antigen/IgE immune complex via IgE antibody receptor Fc ε RI and Fc ε RII, discharge pro-inflammatory cytokine, protease and vasoactive lipid medium (such as, leukotriene, Prostaglandin D2 and platelet activating factor) by the stimulation of eosinophilic granulocyte, mastocyte, basophilic granulocyte and other cells confirmed;

Iv) adaptability cell effect, as partially by create antagonism former, antigen/IgE antibody immune complex or with the peptide of the antigen of MHC compound be specific T cell confirm;

V) the Th1/Tc1 immunoreation produced in response to the challenge carried out with antigen/IgE antibody immune complex, as such as by produce to tumor antigen and tumor be specific CD8IFN γ positive T cell confirm;

Vi) humoral response, as novel, the endogenous antibody by create antagonism antigen or antigen/IgE immune complex confirm.

As used herein, " effective dose " of IgE monoclonal antibody of the present invention is a kind of like this amount, and it is enough to the epi-position of the TAA identifying and be combined into cell surface antigen according to the present invention, and induces, causes or strengthen reference immunoreation.

The present invention also provides the induction IgE immunoreactive method of mediation, and this immunoreation antagonism can produce the cell surface antigen on the circulating tumor cell in the subject of this reaction.The method comprises the IgE monoclonal antibody of the epi-position using the specific binding circulating tumor cell surface of effective dose to experimenter, thus causes the immunoreation to antineoplastic IgE mediation.

As used herein, the immunoreactive inducing moiety of the IgE mediation triggered by circulating tumor cell or the cell surface antigen clinically in unconspicuous transfer by following any one confirmed:

I) completely or partially because of Tumor suppression growth and/or promotion tumor destruction to the marrow source sexual cell reprogrammed in tumor microenvironment;

Ii) acute inflammation occurred in tumor environment via the effector lymphocyte with people Fc epsilon receptor and follow-up Tumor growth inhibition and/or destruction, described receptor can guide ADCC immunoreation to the antigen in microenvironment, ADCP immunoreation or ADCC and ADCP two kinds of immunoreation in conjunction with monoclonal IgE antibody;

Iii) by there is the Secondary cases t cell responses that T cell confirms, thus cause Tumor growth inhibition or cracking and destruction, described T cell shows the specificity to target tumor antigen or extra (secondarilyadditional) antigen of secondary, and described antigen is derived from tumor and expresses under the background of MHC on tumor cell; Or

Iv) by the antitumor t cell responses that the T cell producing other antigens anti-confirms, other antigens described are relevant to the tumor cell that such as there occurs cracking above in (ii).

The present invention also provides induction to the TAA on constitutional tumor surface in experimenter or the ADCC immunoreation mediated the direct IgE of the TAA on loop jump oncocyte surface, ADCP immunoreation or ADCC and ADCP two kinds of immunoreactive methods, described experimenter can produce this immunoreation, described method comprises: the IgE monoclonal antibody using the single epi-position of the specific binding circulating antigen of effective dose to experimenter, the ADCC immunoreation wherein causing the IgE for this antigen to mediate and possible or optional ADCP immunoreation.In a preferred embodiment, ADCC immunoreation and may or optional ADCP immunoreation cause in the microenvironment of tumor or tumor cell.In another embodiment, ADCC immunoreation and may or optional ADCP immunoreation can cause the cracking of the tumor cell in tumor microenvironment via onlooker, secondary effect (bystander, colateraleffect) and kill and wound or tumor cell cracking and kill and wound.

The present invention also provides the method for the cancer that treatment is relevant to the antigen of antibody specificity of the present invention, and mode is the compositions of the IgE monoclonal antibody using at least single epi-position comprising specific binding tumor associated antigen of the present invention.Such cancer includes but not limited to cancer of pancreas, gastric cancer (cancer of stomach), colorectal cancer and pulmonary carcinoma.The cancer of the other types for the treatment of by method of the present invention includes but not limited to: osteosarcoma, esophageal carcinoma, pulmonary carcinoma, mesothelioma, hepatocarcinoma, gastric cancer, cancer of pancreas, colorectal cancer, rectal cancer, colon cancer, tumor of ureter, cerebroma, carcinoma of gallbladder, cholangioma, cancer of biliary duct, renal carcinoma, breast carcinoma, bladder cancer, ovarian cancer, cervical cancer, carcinoma of prostate, thyroid carcinoma, tumor of testis, Kaposi's sarcoma, oberkieferkrebs, carcinoma of tongue, lip cancer, oral cancer, laryngeal carcinoma, laryngocarcinoma, myosarcoma, skin carcinoma etc.

In one embodiment, the invention provides the therapeutic IgE monoclonal antibody of the epi-position by using specific binding MUC1 of the present invention to experimenter and treat the method for the epithelium source cancer in experimenter.In a preferred embodiment, the therapeutic IgE monoclonal antibody of the single epi-position in conjunction with MUC1 of the present invention by suppressing the tumor microenvironment reprogrammed in primary tumor or prophylaxis of tumours transfer, or promotes tumor to grow to the foundation of the network of the host source myeloid cell outside its tissue of origin by interference and prevents clinically or the foundation that radiograph significantly shifts.

In one embodiment, term " to tumor microenvironment reprogrammed " means host in tumor and settles down in source myeloid cell or be involved in IgE that antigen on the irritated effector lymphocyte in tumor combines subsequently to the net effect of growth and metastasis of tumours.Once be attached to be arranged in tumor microenvironment region antigen and host's source cell on high-affinity Fc ε RI, namely tumor behavior changes, and makes tumor to form transfer, as by symptom, health check-up or radiophotography confirm.In one embodiment, tumor microenvironment reprogrammed is promoted by forming ternary complex.In one embodiment, ternary complex is made up of IgE antibody, effector lymphocyte and tumor associated antigen.

The present invention also provides pharmaceutical composition.Such compositions comprises the antibody of the present invention for the treatment of effective dose and pharmaceutically acceptable supporting agent.In a preferred embodiment, pharmaceutical composition comprises the therapeutic IgE monoclonal antibody of the single epi-position of specific binding MUC1 of the present invention.

In one embodiment, term " pharmaceutically acceptable " mean to be ratified by regulator that is federal or state government or in American Pharmacopeia or other generally acknowledged pharmacopeia listed for animal more particularly for the mankind's.Term " supporting agent " refers to diluent, adjuvant, excipient or the vehicle used together with therapeutic agent.Such pharmaceutical carriers can be sterile liquid, such as water and oil, comprises those oil that oil, animal, vegetable or synthesis are originated, such as Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods.When pharmaceutical composition is used through intravenous, water is preferred supporting agent.Saline solution and dextrose and glycerine water solution also can be used as liquid carrier, in particular for injection solution.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glycerol monostearate, Talcum, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol etc.If needed, compositions can also comprise wetting agent or emulsifying agent or the pH buffer agent of trace.These compositionss can be the form of solution, suspension, emulsion, tablet, pill, capsule, powder, slow releasing preparation etc.Compositions is mixed with suppository by traditional binding agent and supporting agent such as triglyceride.Oral formulations can comprise standard supporting agent, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.Described by having in " Remington'sPharmaceuticalSciences " that the example of suitable pharmaceutical carriers is shown at E.W.Martin.Such compositions will comprise antibody or its fragment for the treatment of effective dose, the form preferably in purification, and appropriate supporting agent is to provide the form being correctly administered to patient.Preparation should be applicable to method of application.

According to method of the present invention, the compositions comprising IgE monoclonal antibody of the present invention is used by approach suitable in any immunology.Such as, by intravenous, subcutaneous, intraperitoneal, sheath, antibody introduces in patient body by intravesical, Intradermal, intramuscular or intralymphatic approach.Compositions can be the form of solution, tablet, aerosol or heterogeneous agent.Liposome, long circulating liposomes, immunoliposome, Biodegradable microspheres, micelle etc. also can be used as supporting agent, vehicle or delivery system.In addition, use in vitro program well known in the art, blood or serum can be extracted in patient body; Optionally, preferably purification may be carried out to the antigen in blood samples of patients; Then blood or serum can be mixed with the compositions comprised according to bonding agent of the present invention; Finally treated blood or serum are refilled in patient body.The present invention should not be limited to any specific method introduced by bonding agent in patient body.

In a preferred embodiment, according to conventional program, compositions is mixed with the pharmaceutical composition being suitable for being administered to the mankind through intravenous.Usually, the compositions used for intravenous is the solution in sterile isotonic aqueous buffer.If compositions will be used by infusion, then its available infusion bottle containing sterile pharmaceutical grade water or saline distributes.If compositions is used by injecting, then can provide ampoule of sterile water for injection or a saline, can mix composition before administration.

Compositions of the present invention can be formulated into neutrality or salt form.Pharmaceutically acceptable salt comprise formed with anion those and formed with cation those, described anion is such as derived from those of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., and described cation is such as derived from those of the hydroxide, 2-aminopropane., triethylamine, 2-ethylaminoethanol, histidine, procaine etc. of sodium, potassium, ammonium, calcium, ferrum.

Compositions of the present invention is determined effectively treating, suppressing the amount occurring to shift with the prevention tumor relevant to the antigen of antibody specificity of the present invention by standard clinical techniques.The exist standard skin welt-flare reaction that situation by intradermal administration in response to purifying antigen (such as MUC1) produce of IgE antibody in extravascular compartments measures.In addition, optionally adopt vitro assay to help to determine best dosage range.The exact dose be ready to use in preparation also will depend on the seriousness of route of administration and disease or obstacle, and should decide according to the situation of the judgement of practitioner and each patient.Effective dose can obtain from the dose-effect curve extrapolation deriving from external or animal model test system.

For antibody of the present invention, the dosage being administered to patient is generally 0.001 μ g/kg to 1mg/kg weight in patients.Preferably, be administered to the dosage of patient between 0.01 μ g/kg and 0.1mg/kg weight in patients, more preferably 0.02 μ g/kg to 20 μ g/kg weight in patients.In general, IgE monoclonal antibody of the present invention has much higher Fc ε R affinity (such as compared with IgG antibody) and longer people's Half-life in vivo than the antibody deriving from other species.Therefore, usually can adopt the antibody of the present invention of more low dosage and more low-frequencyly to use.

Pharmaceutical composition of the present invention also can be used in conjoint therapy, namely with other medicament couplings.Such as, conjoint therapy can comprise compositions of the present invention and other antitumor agents of at least one, efficacy enhancing agent and/or safety reinforcing agent.

Pharmaceutical composition of the present invention has in vitro and in vivo Treatment and diagnosis function.Such as, these molecules can be administered to the cell in cultivation, such as external or in vitro, or the cell in subject, such as, in body, with Therapeutic cancer.As used herein, term " experimenter " is intended to comprise the mankind and non-human animal.Preferred experimenter is the human patients suffering from cancer.As used herein, term " treatment " cancer comprises: occur neoplasm metastasis in prevention patient; Suppress the generation of cancer in patient; The tumor load of preexist in the patient eliminated or reduce the cancer suffering from metastatic carcinoma or be confined to primitive organ; Extend the catabasis of cancer patient after passing through chemotherapy and/or operation Primary treatment; And/or any cycle extended in patient between cancer remission and cancer return.

As used herein, " suppression " in the context of the present invention means to slow down, hinder, limit, reduce or prevent.Such as, mean to slow down, hinder, limit, reduce or prevent primary tumors cells to shift when term " transfer of suppression primary tumors cells " uses in this article.

As used herein, " using " refers to any action causing the compositions comprising antibody of the present invention being exposed to predetermined cell or tissue (being generally mammal) or contacting with it.As used herein, use can in vivo, external or carry out in vitro.Such as, compositions is by injecting or being used by endoscope.Use also to comprise and apply according to compositions of the present invention directly to cell.Such as, in operation process, tumor cell can be exposed.According to one embodiment of the invention, these cells (or tumor) exposed can directly be exposed to compositions of the present invention, such as, by washing or pour into operative site and/or cell, or by directly at intra-tumoral injection IgE product itself.

When being used for the treatment of the therapy of cancer, (namely antibody of the present invention to treat effective dose, treatment is obvious tumor clinically, or occurs the amount clinically needed for obvious tumor at initial site or prevention at a distance at certain time point in the future) be administered to patient.Antibody of the present invention and the pharmaceutical composition comprising described antibody through parenteral administration (when it is possible), or will be used at target cell position usually, or use through intravenous.

In another embodiment, IgE antibody of the present invention can be used jointly with the second therapeutic agent (such as, chemotherapeutics).Such therapeutic agent comprises such as antitumor agent, such as doxorubicin, cisplatin, Bleomycin Sulphate, paclitaxel, carmustine, chlorambucil and cyclophosphamide.These medicaments itself only to have patient in toxicity or subtoxic level effectively.In addition, do not think that these medicaments to be diffused in the common solid tumors outside its tissue of origin effectively.By jointly using tumour-specific IgE antibody and common chemotherapeutics, target will be the activity being strengthened chemotherapeutics by the appearance of Prophylactic chemotherapy drug resistance, and chemotherapy resistance is the result of the host source non-tumor cell in microenvironment.

Pharmaceutical composition of the present invention can comprise one or more and be selected from following other chemotherapeutics: chlormethine (such as cyclophosphamide and ifosfamide), aziridine (such as thiophene is for group), alkylsulfonate (such as busulfan), nitroso ureas (such as carmustine and streptozotocin), platinum complex (such as carboplatin and cisplatin), atypia alkylating agent (such as dacarbazine and temozolomide), folate analog (such as methotrexate), purine analogue (such as fludarabine and purinethol), neplanocin (such as cladribine and pentostatin), pyrimidine analogue (such as fluorouracil (being combined individually or with folinic acid) and gemcitabine), the urea (such as hydroxyurea) replaced, antitumor antibiotics (such as bleomycin and doxorubicin), epipodophyllotoxin (such as etoposide and teniposide), microtubule agent (such as docetaxel and paclitaxel), camptothecin analogues (such as irinotecan and hycamtin), enzyme (such as asparaginase), cytokine (such as interleukin-2 and interferon-' alpha '), monoclonal antibody (such as trastuzumab and bevacizumab), recombinant toxin and immunotoxin (such as recombinant cholera toxin-B and TP-38), cancer gene therapy, physiotherapy (such as thermotherapy, radiotherapy and operation) and cancer vaccine (such as the vaccine of anti-telomerase).

By following non-limiting example, the present invention is illustrated further.

Embodiment

Embodiment 1: form chimeric IgE genophore

Hybridoma VU-3C6 and VU-4H5 is produced for two kinds of people's MUC-1 (hMUC-1) (being derived from the mucin of process LAN in epithelioglandular tumor) different isotypes.Check order from each hybridoma clonal antibody variable gene segment, then use standardization program to be grafted on people k light chain and ε heavy chain gene segment.This sequence and database sequence (such as GenBank:J00241.1) are then compared by the kcDNA clone deriving from human peripheral lymphocyte.Clone ε constant region cDNA from the hybridoma (SKO-007, ATCCCRL8033-1) of expressing IgE, then compare with the genome sequence (such as GenBank:J00222.1) in data base.By final IgE mouse-human chimeric antibody called after 3C6.hIgE and 4H5.hIgE.Final plasmid illustrates in figure ia.

1F5 hybridoma targeted human CD20 (hCD20), this is the critical treatment target of a kind of pan-B cell sign thing and treatment B cell lymphoma and various autoimmune disease.From 1F5 hybridoma clonal antibody variable gene segment, and standardization program is used to be grafted on people Igk light chain and ε heavy chain gene as mentioned above.By final IgE chimeric antibody called after 1F5.hIgE.Final plasmid illustrates in figure ia.

Final plasmid transfection is entered CHO-K1 cell (American type culture collection (ATCC), Manassas, VA) to prepare and antibody purification.Use anti-hIgE affinity column, by people IgE by fast protein liquid chromatography (FPLC) purification.The SDS-PAGE of chimeric IgE and people IgE isotype controls (deriving from the IgE of human myeloma SKO-007, in a similar fashion purification) that Figure 1B shows purification compares.All three chimeric antibody samples are all pure, have faint difference in size (may be due to different glycosylation patterns) compared with contrast hIgE.Under the reducing conditions, ε heavy chain moves at 75kDa, is different from the gamma heavy chain moved as 50kDa polypeptide.

Test the ability of its corresponding native antigen (that is, CD20 and MUC1) of antibodies of purification.As via flow cytometry analyzed, 1F5.hIgE in combination with the A20 mouse B cell lymphoma of h CD20 transfection, but not in conjunction with wild-type cell system (the left figure of Fig. 1 C).Similarly, 3C6.hIgE is attached to the 4T1 Mouse mammary cells by people MUC1 transfection, but is not joined to the 4T1 cell (scheming in Fig. 1 C) of untransfected.4H5.hIgE be attached to be derived from people MUC-1 tandem sequence repeats, ectodomain 50-mer peptide, as by ELISA detect, but not in conjunction with control peptide (the right figure of Fig. 1 C).Therefore, described people ε and k constant region and preparation and purification schemes is used not to affect the identification of variable region to antigen of original mouse antibody.

The IgE mediate tumor cytotoxicity that embodiment 2:1F5.hIgE (anti-hCD20) produces

In order to determine whether anti-hCD20 chimeric antibody 1F5.hIgE has functional IgEFc district, we have studied the ability that it activates umbilical cord haematogenous mastocyte (CBMC).Use the measuring of activating as CBMC of generation of interleukin-8 (IL-8), we observe: the CBMC wrapping quilt with 1F5.hIgE in advance when there is the mouse B cell (A20.hCD20) of hCD20 transfection but not the cell of untransfected create IL-8.Similarly, only have and wrap the CBMC of quilt just to hCD20 with anti-hCD20IgE (1F5.hIgE) ⁺human B cell OCI-Ly8 produces reaction, and does not produce IL-8 under these conditions with the CBMC that quilt is wrapped in contrast IgE (SKO).These data show that 1F5.hIgE activates mastocyte with antigen dependency and antigen-specific fashion.

The cytotoxicity of mastocyte and IgE mediation

In order to test the potential cytotoxic effect that tumour-specific IgE mediates, we pay close attention to known expression high-cutting slope along road Fc ε RI and the effector lymphocyte reacted to this receptor.Research worker has before reported the cytotoxicity (Karagiannisetal. (2003) .EurJImmunol33:1030-1040) mediated by person monocytic cell and tumour-specific IgE.We use U937 mononuclear cell to observed similar result in the system of oneself.In this report, we pay close attention to the data of end user's mastocyte and eosinophilic granulocyte's acquisition; These two kinds of cell types relate to the morbidity and tissue injury (Rothenbergetal., 2006NatRevImmunol8:205-217 observed in allergy and asthma; GouldandSutton.2008NatRevImmunol8:205-217; Tsaietal., 2005ChemImmunolAllergy87:179-197).Mastocyte especially receives publicity, produce blastomogenic tissue because they are positioned at, express high-caliber Fc ε RI and trigger the inflammatory reaction coordinated when activating, this reaction raise may mediate tumor disappear eosinophilic granulocyte, neutrophil cell and other effector lymphocytes (TheoharidesandConti, 2004TrendsImmunol25:235-241).Before, confirmed that mastocyte plays via TNF and peroxidase system and killed function of tumor (Hendersonetal., 1981JExpMed153:520-533; Benyonetal., 1991JImmunol147:2253-2258; Ozdemir, O.2007JImmunolMethods319:98-103).

In order to whether the mastocyte testing activation can direct inducing death of neoplastic cells, we prepared purification, mastocyte through cultivating by they and IgE and tumor incubation.The mastocyte (CBMC) through cultivating being derived from Cord blood is functionally similar to from the existing human mast cell (Saitoetal., 1996JImmunol157:343-350) be separated of tissue.CBMC and OCI-Ly8B cell is mixed when there is anti-CD20 (1F5.hIgE) or contrast (SKO-007) IgE.After 24h, add propidium iodide (PI) with labelling dead cell, and by flow cytometry, mixture is analyzed.Also be PI ⁺cFSE ⁺the percentage ratio of cell represents point rate of the death/dying tumor cell of existence.When adding 1F5.hIgE, compared with control antibodies, observed the increase (Fig. 2 A) of cytotoxicity.By effector lymphocyte: target cell ratio increases to the amplitude that more than 2:1 does not increase this effect.

In order to research mechanism, algoscopy (Karagiannis2003supra) is engulfed by the antibody dependent cellular announced, mastocyte is used c-kit antibody labeling, and measure the percentage ratio of c-kit+/CFSE+ cell when there is specificity or contrast IgE.We do not observe any obvious IgE dependency activate the phagocytic capacity (CBMC by this activity can inducing death of neoplastic cells) of mastocyte, and cell mixture and a series of blocking antibody or rabbit igg 1 isotype controls are carried out incubation.Add anti-TNF and cytotoxicity is down to 14.0 ± 0.3% (Fig. 2 B) from 24.2 ± 3.5%.For other tested antibody, seen slight reduction, but these differences are without statistical significance.

The cytotoxicity of eosinophilic granulocyte and IgE mediation

Tumor eosinophilia be associated with prognosis bona, especially (Fernandez-Aceneroetal., 2000Cancer88:1544-1548 in gastroenteric tumor; Iwasakietal., 1986Cancer58:1321-1327; Pretlowetal., 1983CancerRes43:2997-3000).

Before, the people such as Karagiannis reported from human peripheral be separated eosinophilic granulocyte when testing with ovarian cancer cell line IGROV with IgE dependency mode mediating cytotoxicity (Karagiannisetal., 2007JImmunol179:2832-2843).In order to obtain the natural mankind eosinophilic granulocyte of sufficient amount, we break up Cord blood mononuclear cells when there is IL-3 and IL-5.The eosinophilic granulocyte obtained by the program is shown and the phenotype of peripheral blood eosinophilic granulocyte and functional similarity (Zardinietal., 1997JImmunolMethods205:1-9).After 3 weeks, as by flow cytometry analyzed, in culture, the living cells of >95% is similar to ripe eosinophilic granulocyte (CD66b in phenotype ⁺, CD16 ^-).We are by these cell called after umbilical cords haematogenous eosinophilic granulocyte (CBEo).We are by CBEo and OCI-Ly8B mixing with cells, and with the addition of contrast (SKO) or tumour-specific (1F5.hIgE) IgE antibody of 5.0 μ g/ml.At 37 DEG C after 24h, add PI with labelling dead cell, and by flow cytometry, mixture is analyzed (Fig. 2 C).With observed by CBMC the same, CBEo triggers the increase of death of neoplastic cells when there is tumour-specific IgE compared with contrast IgE.Interestingly, the antibody dependent of this effect is not too obvious under higher effector lymphocyte-target cell ratio, thus shows that higher eosinophilic granulocyte and tumor ratio can cause cell death by Antibody independent mode.

In order to study the mechanism of cytotoxicity of CBEo mediation, culture and one group of blocking antibody and inhibitor are carried out incubation (Fig. 2 D) by us.When we observe the blocking antibody adopting TNF related apoptosis-inducing ligand (TRAIL), the appropriateness of tumor mortality reduces, and observed more obvious effect when adding heparin (10U/ml) of low concentration.Think heparin (a kind of anion molecule) pass through in and eosinophilic granulocyte release cationic protein (eosinophile cationic protein (ECP), major basic protein (MBP), eosinophile peroxidase (EPO) and eosinophilic granulocyte source neurotoxin (EDN)) and play this effect (Swaminathanetal., 2005Biochemistry44:14152-14158).Confirm, these cationic proteins cause eukaryotic cell death (Carrerasetal., 2003Biochemistry42:6636-6644) by destroying electronegative cell membrane.

The Cytokine expression profile of the CBMC activated

A large amount of genes is raised (Sayamaetal., 2002Immunol3:5) by when IgE and antigenic activation by the mastocyte activated at mastocyte.Produced by the mastocyte activated by tumour-specific IgE to study which cytokine/chemotactic factor, we have carried out without inclined examination (unbiasedscreen) cytokine.Use based on the Multiplex assays of microballon, to derive from by with IgE and tumor cell Dual culture and the supernatant of mastocyte that activates analyzes 36 kinds of cytokines.By following two kinds of methods, CBMC is activated 24 hours by Fc ε RI at 37 DEG C: with IgE and anti-IgE Dual culture, or by the tumor cell Dual culture by anti-hCD20IgE and expression hCD20.Have evaluated one group of inflammatory, growth and chemotactic factor.Activating and the obvious cytokine raised in tranquillization mastocyte to identify, we apply 2 class chip significance analysis (SAM) algorithms (q<0.05, multiple change >5.0).SAM identifies inflammatory cytokine, such as macrophage inflammatory protein (MIP)-1 α, MIP-1 β, granulocyte-macrophage colony stimutaing factor (GM-CSF), epithelium neutrophil activation peptide 78 (ENA78) and IL-8.As expection, the response strength when mastocyte is activated by polyvalent antigen (such as, cell surface antigen) is greater than response strength when being activated by simple bivalence crosslinked (the anti-IgE of IgE+).

Embodiment 3: anti-MUC1IgE antibody suppression tumor growth in vivo

In order to the anti-tumor in vivo testing anti-hMUC1IgE (3C6.hIgE) is active, we define the mouse cell line of the transmembrane of expressing hMUC1.Total length hMUC1cDNA transfection is entered mammary carcinoma 4T1 by us.The breast carcinoma of 4T1 spontaneous generation from Balb/c mice is separated (Dexteretal., 1978CancerRes38:3174-3181), and be used as the transplantation model (Pulaskietal., 2001CurrProtocImmunolChapter20:Unit2022) of breast carcinoma.The hMUC1 of 4T1.hMUC1 expressed in abundance on its cell surface, and subcutaneous to be similar to the viewed Growth kinetics of parental generation 4T1 cell line in hFc ε RI transgenic mice.

People Fc ε RI mouse model

Acting on in the body of chimeric people's IgE antibody target tumor to study, we used people Fc ε RI α transgenic mice (hFc ε RITg ⁺).In these mices, the endogenous gene of the α-subunit of coding high-cutting slope along road Fc ε RI α is destroyed, and mice is human homology's thing genetically modified (Dombrowiczetal., 1996JImmunol157:1645-1651) under the control of people Fc ε RI α promoter.Be limited to the wild-type mice of mastocyte and basophilic granulocyte by contrast with Fc ε RI alpha expression, Fc ε RI α is at hFc ε RITg ⁺expression scope in mice is similar to the scope seen in the mankind.Except mastocyte and basophilic granulocyte, at hFc ε RITg ⁺in mice (and mankind), Fc ε RI also expresses (Kinet, J.P.1999AnnuRevImmunol17:931-972 on eosinophilic granulocyte, mononuclear cell, Langerhans cell, B cell and eosinophilic granulocyte; Kayabaetal., 2001JImmunol167:995-1003).HFc ε RI α gene outcome has with mice β and γ subunit compound to form functional 4 chain receptors (α β γ ₂) ability.HFc ε RITg ⁺mice produces anaphylaxis (Dombrowiczetal., 1996supra) to human IgE antibodies and anaphylactogen.

In order to verify the ability that these mices are reacted to people IgE, 4T1.hMUC1 tumor cell was administered in peritoneum by we, then used contrast IgE (being derived from SKO-007) or anti-hMUC1 people IgE (3C6.hIgE) at the 9th day.After 24h, perform peritoneal lavage, collecting cell, prepares cell centrifugation smear, then uses hematoxylin, Yihong and Toluidine blue staining.The mastocyte deriving from matched group is intact, and those cells deriving from anti-hMUC1 group demonstrate obvious threshing evidence.This shows to derive from hFc ε RITg ⁺the mastocyte of mice can be reacted to people IgE with antigen-specific fashion.

Tumour-specific IgE suppresses tumor growth in vivo

At hFc ε RITg ⁺the ability that hMUC1 specific IgE affects 4T1.hMUC1 tumor growth is in vivo tested in mice.For these experiments, we consider intravenous and the intraperitoneal delivery of IgE.We find, IgE is removed in vivo fast.Make us at cancer week regional administration together with the fact of the not good vascularization of this observation and Subcutaneous tumor.

By 4T1.hMUC1 tumor cell (altogether 10 ⁵individual) subcutaneous (s.c.) be inoculated into mice shave mao both sides, and carry out processing (Fig. 3 A) at the 1st, 2,3,4 and 5 day with 20 μ gSKO or 3C6.hIgE.We observed the Tumor growth inhibition (tumor size reduces 24%, and two-way ANOVA shows p<0.001) of appropriateness in the mice with 3C6.hIgE process.In addition, in matched group, 8 mices only have 3 to survive by the 34th day, and have 5 still to live in 8 mices in anti-hMUC1 group.

Obtain tumor sample the 34th day mice from survival, and use toluidine blue to carry out dye (Fig. 3 C) for the existence of mastocyte.We have detected the existence of mastocyte in tumor week and tumor in the tumor of the mice of contrast IgE process of must using by oneself.Except a mice, do not observe the remarkable increase of mastocyte or number of inflammatory cells in the mice of 3C6.hIgE process, a described mice has mast cells infiltration, with the evidence of mastocyte threshing in a tumor biopsy.Even with anti-hMUC1IgE (3C6.hIgE) process after, tumor also maintain hMUC1 express, as by immunohistochemistry analyzed.

Tumor response is strengthened by local delivery tumour-specific IgE and cytokine

In our In vivo model, there is not the reaction of obvious antitumor may be due to following two factors: the first, can not by enough antibody delivery to the bad and Subcutaneous tumor that is that grow fast of vascularization; The second, in the microenvironment of these transplantation tumors, lack enough effector lymphocytes.In order to test these variablees, we define the 4T1 cell line (table 1) producing anti-hMUC1 mice IgE (mIgE) or chemotactic cytokine MCP-1 and IL5.

Table 1:4T1 derived cell system

^arecord in mice IgE specific ELISA assay

^bcatch in ELISA algoscopy at mouse cytokine specificity and record

Due to following report, we select MCP-1/CCL2: this cytokine to be produced in response to oncogene activation by tumor cell and can be responsible for mononuclear cell and mastocyte chemotactic enters tumor Zhou Jizhi (Souceketal., 2007NatMed13:1211-1218).Interleukin-15 is somatomedin and the chemotactic factor of eosinophilic granulocyte (Sanderson, C.J.1988DevBiolStand69:23-29).We are by producing the combination of the 4T1 cell of antigen/cytokine and the 4T1 mixing with cells of producing mIgE, then by they subcutaneous injection hFc ε RITg ⁺mice (Fig. 4 A).Produce antibody and the mixture of the tumor of antigen expressed to grow in a progressive way, and express MCP-1 and IL-5 but to lack the tumor that local antibody produces the same.By contrast, what the tumor expressing anti-hMUC1 mice IgE, target antigen and two kinds of cytokines was failed in 8 mices 7 to merely hit growth (Fig. 4 B).In product this single mice blastomogenic, tumor only starts visible the 19th day instead of the 8th day.The immune infiltration thing containing eosinophilic granulocyte is observed in the tumor expressing MCP-1 and IL5.But, only just observe oncolysis when also there is tumour-specific IgE.These data show: when enough tumour-specific IgE being delivered to the tumor with antigen when there is irritated effector lymphocyte, can be observed complete and lasting reaction.

Interestingly, when the opposition side of 7 mices of its tumor has been repelled in wild type 4T1 tumor cell (neither produce hIgE and also do not produce chemotactic factor) subcutaneous injection, wild type tumor has failed to produce after 30 days (data are not shown).These data show that the tumor cell of IgE+ chemotactic factor transfection can stimulate adaptability t cell responses, and preventability ground and the tumor vaccine being indirectly used as stimulation anamnestic response.

In order to confirm this significant observation result, be repeated this experiment, and result (C schemes, each tumor, and D schemes, the meansigma methods of gross tumor volume) shown in Figure 5.In the 4th group of mice, observed a tumor, its compared with other tumor group with the Growth kinetics greatly reduced.Repeat in experiment at this, we observe the mice with the 3rd group of tumor (only antigen+cytokine) and significantly shift sign creating in early days of Tumor Growth.We infer: the growth fraction the 1st of the tumor implanted in the 3rd group or 2 groups are slightly slow, because the mice of growth tumor is ill, and losing weight in early days at experimentation.

Embodiment 4:IgE antibody inhibiting tumor shifts

People Fc ε RI α transgenic mice described is in embodiment 3 replaced to be repeated the experiment described in Fig. 4 of embodiment 3 with wild-type mice (Balb/c).Carry out this experiment with assessment Fc ε RI express spectra contribution in the viewed tumor response of mediation compared with wild-type mice in transgenic mice.

When repeating the experiment described in embodiment 3 in wild-type mice, we observed different results.Be not the complete tumor rejection (Fig. 4 B and Fig. 5) of the 4th group of tumor seen in transgenic mice, on the contrary, there is (Fig. 6) in the very little tumor that growth kinetics reduces greatly in the 4th group of wild-type mice.Three Fc ε RI α transgenic mices are comprised in this experiment as positive control.Implant in three Tg+ mices of the 4th group of tumor and do not have one (0/3) to demonstrate tumor growth (Fig. 6 D,--).

Even if the 4th group of tumor in wild-type mice is with the Growth kinetics greatly reduced, they also still grow in a stepwise fashion.As we are viewed in experiment before employing Tg+ mice, the time very early in experimentation of the 3rd group of tumor in wild-type mice there occurs transfer.3rd group of wild-type mice body weight alleviates, produce ascites and occur perpendicular hair, the sign of the tumor of extensively distribution in multiple organ and position that all is all.But, in the 4th group of wild-type mice, growing even if they create with the 1st, 2 and 3 group and occurring delayed little tumor, but these mices do not produce as at the sign with the metastatic disease seen in the mice of the 3rd group of tumor.That is, even if tumor exists under all mouse skins, but cytokine MCP-1/IL-5 on tumor behavior (transfer) act on the impact of 3C6-mIgE under be fully reversible with ph change.Follow-up has the mice one month of the 4th group of tumor, and mice does not all produce obvious transfer, but they have the tumor slowly grown in a progressive way on both sides.The result of this Key Experiment gathers in following table 2.

Table 2

MUC1=4T-1/MUC1; The 4T-1 of the specific 3C6 murine heavy chain of MUC1IgE=hMUC1 and light chain transfection; The 4T1/hMUC1 of the cDNA transfection of IL5=mIL5; The 4T1/hMUC1 of the cDNA transfection of MCP-1=MCP-1.

Early stage transfer is all observed in the experiment completed in the Tg+ with the 3rd group of tumor and wild-type mice.This is fully reversible with ph change in the mice with the 4th group of tumor.Difference between Tg+ and wild-type mice is: in Tg+ mice, and the mice of having injected the 4th group of tumor never produces palpable tumor.In wild-type mice, there is growth, although very slow in the 4th group of tumor.Therefore, the effect of mIgE antibody must be carry out reprogrammed to the cell being attracted to tumor by cytokine, and itself does not strictly limit tumor growth.The reasonable dismissal mice in Tg+ mice with the 4th group of tumor not being produced to the reason of transfer is that tumor is eliminated by allergic immune response in injection site.But in wild-type mice, transitivity phenotype is eliminated by mIgE, have nothing to do with the existence of tumor.In other words, only when experiment is carried out in wild-type mice, we just can observe the reversible action (Fig. 6) of immunocytolcine on tumor, and 3C6-mIgE antibody can to the myeloid cell reprogrammed in tumor and around tumor, this may be transplant (in Tg+ mice) by prophylaxis of tumours or greatly slow down its growth (wild-type mice), but all prophylaxis of tumours transfers in both cases.

In addition, the experiment carried out by wild-type mice also shows that antibody can control the tumor of having established, because their growth kineticses in wild-type mice significantly reduce (Fig. 6).

Therefore, a feature of IgE antibody of the present invention is: it can prevent circulating cells to set up necessary microenvironment in liver, lung and skeleton and skin before there is obviously transfer from primary tumor.IgE antibody (being different from IgG antibody) moves out of each tissue in vascular space arrival body.Therefore, IgE antibody will be located uniquely, be formed support the necessary microenvironment of transfer to prevent circulating cells.Therefore undertaken treating by such IgE antibody will prevent significantly transfer to occur and therefore prophylaxis of cancer recurrence.

The patent mentioned herein and scientific literature establish the knowledge that those skilled in the art can obtain.The all United States Patent (USP)s quoted from herein and open or undocumented U.S. Patent application are incorporated to way of reference.The all published foreign patent quoted from herein and patent application are incorporated to way of reference accordingly.Every other disclosed list of references, file, manuscript and the scientific literature quoted from herein are incorporated to way of reference accordingly.

Although specifically show with reference to the preferred embodiments of the invention invention has been and describe, but it will be understood by those of skill in the art that, when not departing from the scope of the present invention that appended claims is contained, the multiple change of form and details can be made in the present invention.It will also be understood that, embodiment as herein described is not mutually exclusive, and can be combined in every way when not departing from the scope of the present invention contained by appended claims.

Sequence table

SEQIDNO:1

<120>3C6.hIgE weight chain variable:

<212>DNA

GCCGCCACCATGTACTTGGGACTGAACTGTGTATTCATAGTTTTTCTCTTAAATGGTGTCCAGAGTGAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAATAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGGGGGGGTACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGTAAGTG

SEQIDNO:2

<120>3C6.hIgE light chain variable:

<212>DNA

GCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTAAGT

SEQIDNO:3

<120>4H5.hIgE monoclonal antibody heavy variable region

<212>DNA

GCCGCCACCATGGGATGGAGCTGTATCATGCTCTTTTTGGTAGCAACAGCAACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTGACTTCAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATCTGCGGACTCTGCGGTCTATTACTGTACAAGGGGGGGTGATTACCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGGTAAGT

SEQIDNO:4

<120>4H5.hIgE monoclonal antibody heavy variable region

<212>DNA

GCCGCCACCATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTAAGT

SEQIDNO:5

The aminoacid sequence of <120>MUC1 epi-position

<212> aminoacid

STAPPAHGVTSAPDTRPAPG

Claims

1. one kind to the method for active reprogrammed of at least one host source non-tumor cell of microenvironment being arranged in patient's solid tumor, the ability that wherein said non-tumor cell mediates described neoplasm metastasis is suppressed, described method comprises the step using the specific IgE antibody of tumor associated antigen, wherein said antibody forms ternary complex in the described microenvironment of described tumor, described ternary complex is by described IgE antibody, described tumor associated antigen and described tumor environment endogenic host source non-tumor cell are formed, wherein said antibody specificity is attached to described tumor associated antigen and is positioned at the antibody receptor on the surface of described host source non-tumor cell, described antibody receptor is that the constant region of described IgE antibody is specific.

2. method according to claim 1, wherein said tumor associated target antigens is expressed on the surface of the cell of described tumor.

3. method according to claim 1, wherein said tumor associated target antigens is soluble antigen.

4. method according to claim 1, wherein said tumor is solid primary tumor or the tumor for transferring to the position different from tumor originates organ.

5. method according to claim 1, wherein IgE antibody is chimeric mAb or Humanized monoclonal antibodies.

6. method according to claim 1, wherein said tumor associated antigen is MUC1, PSA, Her2/neu, CA125 or FBP.

7. method according to claim 1, wherein said IgE antibody is that MUC1 is specific.

8. method according to claim 1, wherein said host source non-tumor cell is myeloid cell.

9. method according to claim 8, wherein said host source non-tumor cell is selected from: mastocyte, basophilic granulocyte, mononuclear cell, macrophage, dendritic cell, Langerhans cell, eosinophilic granulocyte and common Myeloid progenitor cells.

10. method according to claim 1, wherein said host source non-tumor cell is mesenchyme source cell.

11. methods according to claim 10, wherein said host source non-tumor cell is selected from fibroblast, vascular progenitor, endotheliocyte, adipose cell and precursor thereof.

12. methods according to claim 1, wherein said solid tumor is epithelium source tumor.

13. methods according to claim 12, wherein said solid tumor is selected from breast carcinoma, colorectal cancer, ovarian cancer, renal carcinoma, carcinoma of prostate, bladder cancer, human primary gastrointestinal cancers, pulmonary carcinoma and multiple myeloma.

14. method according to claim 1, before wherein shifting in described patient, use described antibody.

15. 1 kinds of methods suppressing the solid tumor in patient to shift, described method comprises uses the IgE antibody that can form ternary complex in the microenvironment of described tumor to described patient, described ternary complex is by the specific described IgE antibody of tumor associated antigen, described tumor associated antigen and described tumor microenvironment endogenic host source non-tumor cell are formed, wherein said antibody specificity is attached to described tumor associated antigen and is positioned at the antibody receptor on the surface of described host source non-tumor cell, described antibody receptor is that the constant region of described IgE antibody is specific, and wherein after the described ternary complex of formation, described non-tumor cell mediation transfer, stimulating growth or mediation are suppressed the ability of the resistance of chemotherapeutics.

16. method according to claim 15, wherein said tumor associated antigen is expressed on the surface of the cell of described tumor.

17. methods according to claim 15, wherein said tumor associated target antigens is soluble antigen.

18. methods according to claim 15, wherein said tumor associated antigen is CA125, FBP (FBP), HER2/neu, MUC1 or PSA.

19. methods according to claim 15, wherein said IgE antibody is that MUC1 is specific.

20. method according to claim 15, before wherein shifting in described patient, use described IgE antibody.

21. 1 kinds of IgE monoclonal antibodies, the epi-position of its specific binding MUC1.

22. IgE antibody according to claim 21, wherein said monoclonal antibody is chimeric mAb or Humanized monoclonal antibodies.

23. IgE antibody according to claim 21, have Ren Yuan constant region.

24. IgE antibody according to claim 21, have Ren Yuan variable region, non-human variable domains or its combination in any.

25. IgE antibody according to claim 21, it combines the epi-position being selected from the MUC1 of SEQIDNO:5.

26. IgE antibody according to claim 21, wherein variable region of heavy chain is by the nucleic acid coding comprising SEQIDNO:1, and wherein variable region of light chain by the nucleic acid coding comprising SEQIDNO:2.

27. IgE antibody according to claim 21, wherein variable region of heavy chain is by the nucleic acid coding comprising SEQIDNO:3, and wherein variable region of light chain by the nucleic acid coding comprising SEQIDNO:4.

28. IgE antibody according to claim 21, are selected from 3C6.hIgE and 4H5.hIgE.

29. 1 kinds of methods slowing down the growth kinetics of patient's solid tumor, comprise the step using the specific IgE antibody of tumor associated antigen, wherein said antibody forms ternary complex in the microenvironment of described tumor, described ternary complex is made up of described IgE antibody, described tumor associated antigen and host source non-tumor cell, wherein said antibody specificity is attached to described tumor associated antigen and is positioned at the antibody receptor on the surface of described non-tumor cell, and described antibody receptor is that IgE is specific.

30. methods according to claim 29, wherein said IgE antibody is that MUC1 is specific.

31. 1 kinds of specific IgE monoclonal antibodies of tumor associated antigen, it is for the ability of the host source non-tumor cell mediation patient solid tumor transfer in Tumor suppression microenvironment.

32. the specific IgE monoclonal antibody of tumor associated antigen, it is for suppressing the neoplasm metastasis of solid tumor in patient.

33. the IgE monoclonal antibody according to any one of claim 31 or 32, wherein said tumor associated target antigens is soluble antigen.

34. IgE monoclonal antibodies according to any one of claim 31 or 32, wherein said tumor associated antigen is MUC1, PSA, Her2/neu, CA125 or FBP.

35. IgE monoclonal antibodies according to any one of claim 31 or 32, wherein said IgE antibody is chimeric mAb or Humanized monoclonal antibodies.

36. the IgE monoclonal antibody according to any one of claim 31 or 32, wherein said IgE antibody is that MUC1 is specific.

37. the IgE monoclonal antibody according to any one of claim 31 or 32, has Ren Yuan constant region.

38. IgE monoclonal antibodies according to any one of claim 31 or 32, have Ren Yuan variable region, non-human variable domains or its combination in any.

39. IgE monoclonal antibodies according to any one of claim 31 or 32, for being selected from the epitope specificity of the MUC1 of SEQIDNO:5.

40. IgE monoclonal antibodies according to any one of claim 31 or 32, wherein variable region of heavy chain is by the nucleic acid coding comprising SEQIDNO:1, and wherein variable region of light chain by the nucleic acid coding comprising SEQIDNO:2.

41. IgE monoclonal antibodies according to any one of claim 31 or 32, wherein variable region of heavy chain is by the nucleic acid coding comprising SEQIDNO:3, and wherein variable region of light chain by the nucleic acid coding comprising SEQIDNO:4.

42. the IgE monoclonal antibody according to any one of claim 31 or 32, is selected from 3C6.hIgE and 4H5.hIgE.

43. IgE monoclonal antibodies according to any one of claim 31 or 32, wherein said solid tumor is selected from breast carcinoma, colorectal cancer, ovarian cancer, renal carcinoma, carcinoma of prostate, bladder cancer, human primary gastrointestinal cancers, pulmonary carcinoma and multiple myeloma.